microorganisms Article The Stable Fly (Stomoxys calcitrans) as a Possible Vector Transmitting Pathogens in Austrian Pig Farms Lukas Schwarz 1,* , Andreas Strauss 1, Igor Loncaric 2 , Joachim Spergser 2, Angelika Auer 3, Till Rümenapf 3 and Andrea Ladinig 1 1 Department for Farm Animals and Veterinary Public Health, University Clinic for Swine, University of Veterinary Medicine, 1210 Vienna, Austria; [email protected] (A.S.); [email protected] (A.L.) 2 Department of Pathobiology, Institute of Microbiology, University of Veterinary Medicine, 1210 Vienna, Austria; [email protected] (I.L.); [email protected] (J.S.) 3 Department of Pathobiology, Institute of Virology, University of Veterinary Medicine, 1210 Vienna, Austria; [email protected] (A.A.); [email protected] (T.R.) * Correspondence: [email protected]; Tel.: +43-664602756848 Received: 26 August 2020; Accepted: 24 September 2020; Published: 25 September 2020 Abstract: This pilot study aimed to investigate stable flies from Austrian pig farms for the presence of defined swine pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2), hemotrophic mycoplasmas in ingested blood and/or body parts and bacteria on the surface of the flies. Furthermore, the use of stable flies as a diagnostic matrix for the detection of pathogens in the ingested pig blood should be investigated. In total, 69 different microorganisms could be found on the surface of tested S. calcitrans from 20 different pig farms. Escherichia coli was the most common bacterium and could be found on flies from seven farms. In seven farms, hemotrophic mycoplasmas were detected in stable flies. PRRSV could not be found in any of the samples of these 20 farms but PCV2 was detected in six farms. Whether the stable fly can be used as a matrix to monitor the health status cannot be accurately determined through this study, especially in regard to PRRSV. Nevertheless, it might be possible to use the stable fly as diagnostic material for defined pathogens like Mycoplasma suis and PCV2. Keywords: hemotrophic mycoplasmas; PRRSV; PCV2; bacteria 1. Introduction The stable fly, S. calcitrans, is often found in swine farms. Its significance in food animal production can be divided into direct and indirect influences. Direct influences can include restlessness, pain due to biting, stress, loss of blood, reduced feed intake, lesions of the skin followed by local inflammation and immunosuppression, while indirect effects are due to the transmission of infectious agents [1]. This is supported by Zumpt (1973), who mentioned that the main harming effects caused by stable flies are (i) biting dependent disturbance, (ii) loss of blood and toxic reactions caused by Stomoxys saliva and (iii) the transmission of pathogens [2]. Stable flies feed on blood from their hosts one or two times per day. Usually, they do not finish blood feeding on one animal, since the host animals try to defend against the painful fly bites. Additionally, stable flies can be disturbed by other flies during blood feeding. Both causes for interruption of blood feeding end up in a change of biting site or change of the host [3,4]. S. calcitrans can ingest 3.2–22.6 mg blood [5]. Mellor et al. (1987) investigated the survival of African Swine Fever Virus (ASFV) in stable flies after feeding on infected pigs or after feeding on viremic blood in the laboratory. In this study, it was shown that ASFV can survive or stay infectious in stable flies for up to 48 h [6]. Although the regurgitation of ingested blood by stable flies seems improbable [3], Microorganisms 2020, 8, 1476; doi:10.3390/microorganisms8101476 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 1476 2 of 12 the ingestion of stable flies which fed on ASFV viremic pigs by non-infected pigs was at least a possible way of ASFV transmission under experimental conditions [7]. For other host species, such as horses, stable flies are relevant vectors for transmission of infectious diseases [8–11], but in pigs only a few studies exist [1,6,12]. The objective of this study was to investigate the role of stable flies as a possible vector for swine pathogens under conditions found in Austrian farms. Furthermore, we tried to clarify if S. calcitrans can be used as a diagnostic matrix for the surveillance of defined pathogens in farms where stable flies occur. 2. Materials and Methods As this investigation was a pilot study, we did not calculate any sample size. Stable fly specimens were collected from a total of 20 Austrian pig farms either during routine farm visits or during consultation farm visits performed by the University Clinic for Swine as support for the corresponding herd health veterinarian. Both clinically unsuspicious farms and farms with current health/disease problems were chosen (Table1). Table 1. Overview of the study farms and their clinical status. In total, pigs of five farms were clinically unsuspicious and did not show any clinical symptoms at the time point of sampling. All farms vaccinated piglets against PCV2. Farm Date Case History/Clinical Symptoms PRRS-Status 1 06/2018 Infertile sows Positive, vaccinated 2 06/2018 Clinically unsuspicious Negative, non-vaccinated 3 07/2018 Increased return-to-oestrus rate, vaginal discharge Unknown, non-vaccinated 4 07/2018 Post-partum dysgalactia Negative, non-vaccinated 5 09/2018 Clinically unsuspicious Negative, non-vaccinated 6 09/2018 Enzootic pneumonia Negative, non-vaccinated 7 09/2018 Suckling piglet diarrhoea due to Escherichia coli Negative, non-vaccinated 8 09/2018 Clinically unsuspicious Negative, non-vaccinated 9 09/2018 Clinically unsuspicious Negative, non-vaccinated 10 09/2018 Clinically unsuspicious, PRRS outbreak 10 years ago Unknown, non-vaccinated 11 01/2019 Increased return-to-oestrus rate, chronic bacterial cystitis Negative, non-vaccinated 12 02/2019 Vaginal discharge, vaginitis Positive, vaccinated 13 03/2019 PRRS outbreak and swine dysentery in fattening pigs Positive, vaccinated 14 03/2019 Increased return-to-oestrus rate, weak born piglets Negative, non-vaccinated 15 04/2019 PCV2-reproductive disease Positive, vaccinated 16 04/2019 Glaesserella parasuis-related lameness in sows suspected Negative, non-vaccinated 17 04/2019 PRRS outbreak, eperythrozoonosis Positive, vaccinated 18 06/2019 PRRS outbreak Positive, vaccinated 19 ** 06/2019 Tail biting in fattening pigs due to ascariasis Unknown, non-vaccinated 20 07/2019 PRRS unstable farm Positive, vaccinated PRRS-status: vaccinated = modified live virus vaccines; negative = clinically unsuspicious for any PRRS-related symptoms and no positive PCR/ELISA-test result over the last year. ** Farm 19 was the only fattening farm and all other farms were either piglet-producing farms or farrow-to-finish farms. 2.1. Sampling Stable fly specimens were collected between June 2018 and July 2019. Stable flies were mainly collected in the gestation area, as there the most specimens were observed. Fly specimens were collected from different animals. In each farm, a total of 40 stable flies were caught by hand wearing sterile gloves. Collected flies were placed in 15-mL plastic tubes and kept on crushed ice for transportation to the laboratory and for immobilization. Ten of these 40 fly specimens were used for bacteriological examination. For the virological investigation, another 20 flies were prepared and the remaining 10 flies were stored at 20 C for possible additional or repeated investigations. − ◦ Microorganisms 2020, 8, 1476 3 of 12 2.2. Processing of Flies for Further Analysis For further processing, flies were kept in a refrigerator to cool down to a temperature of about 4 ◦C. In this immobilised stage all flies were prepared for investigations on frozen thermal packs under a laminar flow hood to guarantee a stage of insensitiveness during preparation [12,13]. 2.3. Microbiological Examination For bacteriological examination a pool sample of 10 stable flies was washed in 1 mL physiologic sodium chloride (Kochsalz “Braun” 0,9%—Infusionslösung®, B. Braun Melsungen AG, Melsungen, Germany) solution on a vortex shaker (2 min, 4 ◦C) to get a suspension containing bacteria from the fly surface [14]. After mixing, the suspension was transferred to a 1.5 mL Eppendorf tube and sent to the Institute of Microbiology, University of Veterinary Medicine Vienna, for isolation of cultivable bacteria. For cultivation of bacteria a loop of the washing solution (approximately 10µL) was streaked onto BD Columbia Blood Agar with 5% v/v sheep blood and incubated in aerobic/microaerophilic (5% CO2)/anaerobic conditions, Columbia CNA Improved II agar with 5% v/v sheep blood and BD MacConkey II(all from Becton Dickinson, Germany). Agar plates were incubated at 37 ◦C for 24–48 h. Isolation of fungi was conducted on BD Sabouraud Agar with gentamicin and chloramphenicol (Becton Dickinson, Germany) at 28 ◦C for 3–4 days. After incubation, one colony representing a distinct colony morphotype was identified to species level (if applicable) by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik, Germany). The remaining flies, which had been washed, were used for the detection of mycoplasmas by PCR employing universal primers (hmyc-forward: 50-GGCCCATATTCCTACGGGAAGCAGCAGT-30, hmyc-reverse: 50-TAGTTTGACGGGCGGTGTGTACAAGACCTG-30) for the amplification of the 16S rRNA gene of hemoplasmas and closely related non-hemotrophic mycoplasma species as previously described [15]. Resulting amplicons were sequenced at LGC Genomics, Berlin, Germany, and sequences compared to entries in the GenBank nucleotide database using BLASTN algorithm. 2.4. Virological Investigations All preparation steps were performed on frozen thermal packs ( 20 C). For dissecting, the flies − ◦ were put into a sterile plastic bag onto the thermal pack to keep the flies immobilised and insensitive during the preparation. For virological investigations the mouth parts (proboscis) and the abdominal parts of 20 flies were prepared and pooled using a scalpel [3].